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Viviana M. Berthoud, Eileen M. Westphale, Anna Grigoryeva, Eric C. Beyer; PKC Isoenzymes in the Chicken Lens and TPA-Induced Effects on Intercellular Communication. Invest. Ophthalmol. Vis. Sci. 2000;41(3):850-858.
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purpose. Because lens connexins are phosphoproteins and intercellular
communication between lens cells may be modulated by connexin
phosphorylation, experiments were designed to characterize the
expression of protein kinase C (PKC) isoenzymes in the chicken lens and
in lentoid-containing cultures and to study the effects of
12-O-tetradecanoylphorbol-13-acetate (TPA) treatment on the
distribution of PKC isoenzymes and intercellular communication.
methods. The presence and distribution of PKC isoenzymes were studied by
immunoblot analysis and immunofluorescence in chicken lens sections and
in cell cultures under control conditions and after treatment with TPA.
Intercellular communication was assessed by transfer of microinjected
results. PKC α, γ, ι, ε, and μ were detected in lens homogenates by
immunoblot analysis. The levels of PKC α, γ, ι, and μ decreased
between the 7th and the 18th embryonic days. Levels of PKC ε remained
relatively constant during the period of study. Similarly, lens cells
in culture expressed isoenzymes α, γ, ε, ι, and μ. PKC β
was not detected in lens or culture homogenates. In lens sections, all
PKC isoenzymes analyzed were present in epithelial cells, in the
annular pad region, and in the posterior aspect of fiber cells. The
anti-PKC γ antibody also stained fiber cell membranes. Analysis of
lentoid cultures by immunofluorescence revealed that PKC γ, ε, andι
and minimal amounts of PKC α were present in lentoid cells.
Treatment with 200 nM TPA for 15 to 30 minutes induced translocation of
PKC γ to the plasma membrane of lentoid cells and significantly
reduced the transfer of microinjected Lucifer yellow.
conclusions. Several PKC isoenzymes are expressed by lens cells in situ and in
culture. The γ isoenzyme, present in lens fibers, was activated in
lentoid cells by TPA, a known activator of PKC. We have previously
demonstrated TPA-induced phosphorylation of the gap junction protein
connexin56 (Cx56). The new data presented in the current study
demonstrate that TPA treatment also decreased intercellular
communication. Taken together, the results suggest that differential
phosphorylation of Cx56 by PKCγ may induce a conformational change in
the protein which, in turn, might lead to channel closure.
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